Throughout this application various publications are referred to by Arabic numerals in brackets. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains.
Structural mapping or ‘footprinting’ refers to methods in which the surface of nucleic acids and proteins accessible to the solvent is mapped with as fine as single residue resolution. Footprinting assays can be viewed as a collection of ‘molecular cameras’ that snap pictures of the position of each residue. Footprinting assays examine structure, ligand binding and/or conformational changes by determining the accessibility of the backbone or residues of macromolecules through their sensitivity to chemical or enzymatic modification or cleavage (reviewed by [3, 4]). The key characteristics of a footprinting assay are that i) the reaction of the footprinting probe with the polymer is limited such that each position along the chain is sampled with equal probability, and ii) cleavage or modification products are uniquely identified (FIG. 1).
The ability of proteins to undergo association and folding reactions has long been known to be a fundamental feature of cellular function across all the kingdoms of life. Macromolecular binding and folding reactions critical to unique biological functions are generally referred to as ‘reversible associations’. Among the techniques that have been developed for the study of reversible associations, footprinting occupies a unique niche. Unlike structural methods such as x-ray crystallography and nuclear magnetic resonance (NMR), footprinting achieves site-specific resolution without extensive infrastructure, makes parsimonious use of biological samples and can be performed at dilute concentrations of macromolecules. Footprinting can map static structures as well as equilibrium and time-dependent transitions. The single residue resolution of footprinting can be used to develop detailed models of macromolecular structure, map ligand binding sites and follow conformational changes (FIG. 1). Computational tools are being developed that utilize the ensemble of individual measurements of the residue solvent accessibility to generate structural models of proteins and nucleic acids [6-10].
Cleavage of RNA and DNA by hydroxyl radicals is relatively insensitive to base sequence and whether a nucleic acid is single or double stranded [11, 12]. That hydroxyl radical cleavage of nucleic acids is quantitatively correlated with the solvent accessibility of the phosphodiester backbone has been demonstrated through comparisons of hydroxyl radical footprints with solvent accessibility calculations from crystal structures for protein-DNA complexes [13, 14] and RNA tertiary structures [15-19]. Backbone cleavage of DNA by hydroxyl radical is correlates with the accessible surface of the hydrogen atoms of the nucleotide sugar [20]. Hydroxyl radical footprinting yields a robust and readily interpretable measure of the structure and interactions of nucleic acids (FIG. 1). The availability of modern analysis tools such as CAFA [1] strengthens the feasibility of this method.
Hydroxyl radical footprinting was first extended to proteins by monitoring cleavage of the peptide backbone by gel electrophoresis [21, 22]. However, peptide bond cleavage is inefficient [23]. Thus, further development of protein hydroxyl radical footprinting has focused on the oxidation of amino acid side chains (reviewed in [24] and [25, 26]). Mass spectrometric analysis of proteolytic fragments is used to quantitate the oxidation rate of individual or groups of amino acid side chains. The differential reactivity of the amino acid side chains to oxidation is addressed in thermodynamic and kinetic analyses by quantitating the relative change in residue reactivity [24, 27]. A relationship between hydroxyl radical reactivity and solvent accessibility is emerging for proteins (reviewed in [24] & [25, 26]).
An important virtue of footprinting is that it can provide solution structural information with single residue resolution coupled to thermodynamic and kinetic transitions. Quantitative protocols have been extensively used to determine thermodynamic [3] and kinetic [31-34] constants describing protein-DNA interactions (reviewed in [3]). These protocols have been successfully extended to multiple implementations of quantitative hydroxyl radical footprinting [35-37] (see below). Protocols for thermodynamic protein hydroxyl radical footprinting have been published [38]. The individual-site isotherms [39, 40] and kinetic progress curves [31, 32] determined from thermodynamic and kinetic footprinting studies, respectively, provide an ensemble of local measures of macromolecular transitions from which detailed energetic and mechanistic portraits can be painted [41-44].
Footprinting assays for DNA, RNA and proteins have been developed using a wide range of reagents including the hydroxyl radical (.OH). The hydroxyl radical is among the most reactive and promiscuous of chemical oxidants [45]. Hydroxyl radical can be generated in solution by the Fenton-Haber-Weiss reaction according to the reactionFe(II)-EDTA+H2O2→Fe(III)-EDTA+.OH+OH−  (1).Tullius and co-workers showed that a convenient implementation of this chemistry for footprinting is to reductively cycle Fe(III) back to Fe(II) by the addition of ascorbate [13, 46, 47],
allowing low concentrations (μM) of the iron catalyst in the reaction mixture. This method is widely applied and inexpensive to perform. The reagent concentrations typically used in static and equilibrium .OH footprinting studies are μM in Fe(II)-EDTA and mM in H2O2 and ascorbate with reaction times of several to tens of minutes. Obviously, long reaction times are incompatible with high-throughput implementations.
A method using equation 1 where Fe(II) is stoichiometrically consumed by reaction with H2O2 to produce hydroxyl radicals on the millisecond timescale was recently developed [48-50]. While the reaction time is fast, impediments to high-throughput implementation of equation 1 include the need to precisely add high concentrations of the two reactants and auto oxidation of Fe(II).
Peroxonitrite has been used to hydroxyl radical footprint macromolecules [35, 51]. This reagent has not gained wide acceptance due to limitations on the solution conditions under which the reagent produces significant quantities of hydroxyl radical.
A recently developed method photolyzes H2O2 with UV radiation [25, 26, 52]. The use of UV radiation precludes this approach for DNA and RNA; nucleic acids are damaged by even limited exposure to short wavelength UV light. Thus, a disadvantage of H2O2 photolysis is that it is not general to both proteins and nucleic acids.
Radiolysis of water by ionizing radiation produced either from low flux gamma sources and high flux synchrotron beams has been effectively used to footprint DNA, RNA and proteins [16, 37, 53-56]. The advantage of radiolysis compared to the above described methods is that ‘nothing but light’ is added to the solutions containing the macromolecules to be footprinted. The disadvantage of low flux gamma sources is the need for an expensive gamma source, the long exposure required and cumbersome sample loading and unloading. While high flux synchrotron beams allow very short exposure times, substantial heat may be generated that is intolerable to biological samples. More importantly, the use of a synchrotron requires an application for beamtime and the transport of samples to a remote facility. Therefore, synchrotron footprinting is incompatible with laboratory-based high-throughput implementation.
The past decade has seen the advent of high-throughput structure determination initiatives focused on proteins, DNA and RNA. These initiatives are typically grouped under the rubric ‘structural genomics’. High-throughput structure determination may be particularly valuable in screening studies where large numbers of structures and/or complexes need to be interrogated. Biological function may be revealed only by understanding the acquisition of structure as a function of time or the binding of a ligand requiring the determination of multiple structures for a single reaction.
High throughput structural initiatives have had limited to moderate success despite the infusion of many millions of dollars. While atomic resolution structures are the gold standard for structure determination, biological function can often be gleaned from lower resolution structures. In many cases, the complexity or size of macromolecules and complexes precludes determination of an atomic resolution structure. RNA molecules of even moderate size are notoriously refractory to structure determination.